Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen

JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies.     

Serological and molecular properties of isolates of Bean common mosaic virus-BCMV from Khorasan Razavi province

During growing season, 2011–2012, selected bean plant samples with symptoms of mosaic, vein clearing, leaf rolling, were collected from different part of Khorasan Razavi province of north-eastern Iran. To identify the virus, leaf samples were tested serologically by DAS-ELISA and tissue blot, using specific BCMV polyclonal antibody. Elisa positive samples were rechecked by RT-PCR and IC-RT-PCR using set of primers directed to the coat protein gene which were designed to detect and characterise the viral species. Similarly, the BCMV primers amplified product of approximately 373?bp in 1% agarose gel electrophoresis. In phylogenic analysis, A and B groups were formed for 12 Iranian isolate comparing with 18 BCMV isolates from GenBank. Iranian isolates were classified into three clusters indicating to have more homology 99.3% with Mexican Isolates. There are also indications of some diversities among Iranian BCMV isolates.     

Development of Oligoclonal Nanobodies for Targeting the Tumor-Associated Glycoprotein 72 Antigen

The tumor-associated glycoprotein 72 (TAG-72) is a membrane mucin whose over-expression is correlated with advanced tumor stage and increased invasion and metastasis. In this study, we identified a panel of four nanobodies, single variable domains of dromedary heavy-chain antibodies that specifically recognize the TAG-72 antigen. All selected nanobodies were shown to selectively bind to this cancer-related molecule with low-nanomolar affinities and do not cross-react with other antigens, such as MUC1 or HER2. Furthermore, they can detect TAG-72 in concentrations as low as 5 U/ml which is valuable in sensitive detection of this molecule in cancerous patients. Cell ELISA experiments proved their ability for binding to the native target antigen on TAG-72 expressing cells while not showing any reactivity to HT-29 cells, a TAG-72-negative cell line. Using competition studies, we found that each nanobody recognizes a distinct epitope on the TAG-72 antigen that is different from the one recognized by the mouse anti-TAG-72 antibody, CC49. Considering their high specificity, reduced immunogenicity and multi-targeting behavior, these oligoclonal nanobodies represent a promising tool to target TAG-72 over-expressing tumor cells.     

Deregulation of microRNAs by HIV-1 Vpr protein leads to the development of neurocognitive disorders

Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders.     

The fauna and distribution of house dust mites in residential homes of Bandar Abbas District,Southern Iran

This study was designed to determine the occurrence, distribution and abundance of house dust mites (HDM) in residential homes in Bandar Abbas (Hormozgan Province), because of numerous complaints of allergies in this oriental city. The study area was divided in five sampling zones based on population density and geographical distribution. In each sampling zone 10 houses were randomly selected. A total of 50 home dust samples were collected using a portable vacuum cleaner for 2 min from 1 m² of the surface of mattresses, carpets, sofas and furniture in residential houses. After collection, samples were immediately frozen. Mite species were identified and counted using standard methods and keys. Of the sampled houses 88% (44 houses) were contaminated with at least one HDM species. Three species were identified: Dermatophagoides pteronyssinus (63.1%), D. farinae (32.8%) and D. evansi (4.1%) (Pyroglyphidae). Our findings indicate a relationship between HDM density and moisture and temperature of residential places. The high contamination rate of residential houses (88%) and the favourable environmental conditions for these arthropods stress that they should be considered as important allergic causing agents.     

A human vitamin D receptor mutant activated by cholecalciferol

The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily, involved in calcium and phosphate homeostasis; hence implicated in a number of diseases, such as Rickets and Osteoporosis. This receptor binds 1α,25-dihydroxyvitamin D(3) (also referred to as 1,25(OH)(2)D(3)) and other known ligands, such as lithocholic acid. Specific interactions between the receptor and ligand are crucial for the function and activation of this receptor, as implied by the single point mutation, H305Q, causing symptoms of Type II Rickets. In this work, further understanding of the significant and essential interactions between the ligand and the receptor was deciphered, through a combination of rational and random mutagenesis. A hVDR mutant, H305F, was engineered with increased sensitivity towards lithocholic acid, with an EC(50) value of 10 μM and 40±14 fold activation in mammalian cell assays, while maintaining wild-type activity with 1,25(OH)(2)D(3). Furthermore, via random mutagenesis, a hVDR mutant, H305F/H397Y, was discovered to bind a novel small molecule, cholecalciferol, a precursor in the 1α,25-dihydroxyvitamin D(3) biosynthetic pathway, which does not activate wild-type hVDR. This variant, H305F/H397Y, binds and activates in response to cholecalciferol concentrations as low as 100 nM, with an EC(50) value of 300 nM and 70±11 fold activation in mammalian cell assays. In silico docking analysis of the variant displays a dramatic conformational shift of cholecalciferol in the ligand binding pocket in comparison to the docked analysis of cholecalciferol with wild-type hVDR. This shift is hypothesized to be due to the introduction of two bulkier residues, suggesting that the addition of these bulkier residues introduces molecular interactions between the ligand and receptor, leading to activation with cholecalciferol.     

The Anti-fibrotic Effects of Heat-Killed Akkermansia muciniphila MucT on Liver Fibrosis Markers and Activation of Hepatic Stellate Cells

Probiotics and Antimicrobial Proteins - Hepatic stellate cell (HSC) activation is a key phenomenon in development of liver fibrosis. Recently, Akkermansia muciniphila has been introduced as a...     

Combined TGE-SGE expression of novel PAI-1-resistant t-PA in CHO DG44 cells using orbitally shaking disposable bioreactors

An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncatedmutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncatedmutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.     

Comparison of in vitro antibacterial activities of two cationic peptides CM15 and CM11 against five pathogenic bacteria: Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio cholerae, Acinetobacter baumannii, and Escherichia coli

In recent years, the widespread use of antibiotics has caused many bacterial pathogens resistance to conventional antibiotics. Therefore, generation of new antibiotics to control and reduce the effects of these pathogens is urgently needed. Antimicrobial peptides and proteins are important members of the host defense system in eukaryotes. These peptides are potent, broad-spectrum antibiotics that demonstrate potential as novel and alternative therapeutic agents for the treatment of drug-resistant infections. Accordingly, we evaluated two hybrid peptides CM11 (WKLFKKILKVL-NH2) and CM15 (KWKLFKKIGAVLKVL-NH2) on five important pathogenic bacteria. These peptides are short cecropin?Cmelittin hybrid peptides obtained through a sequence combination approach, which are highly effective to inhibit the growth of important pathogenic bacteria. The activity of these two cationic peptides (CM11 and CM15) in different concentrations (2?C64?mg/L) was investigated against standard and clinical isolates of important hospital infection bacteria by measuring MIC, MBC, and bactericidal assay. These peptides demonstrated the same ranges of inhibitory values: The organisms in early 24?h were more susceptible to polycationic peptides (MIC: 8?mg/L and MBC 32?mg/L), but after 48?h the MIC and MBC remained constant for the CM11 peptide. Bactericidal assay showed that all bacteria strains did not have any growth in agar plates after 40?min. The result showed that these two peptides are more effective than other peptides.     

Real-Time RT-PCR on SAG1 and BAG1 Gene Expression during Stage Conversion in Immunosuppressed Mice Infected with Toxoplasma gondii Tehran Strain

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.